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1.
Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer 总被引:3,自引:1,他引:2
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We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor. 相似文献
2.
Using a 200 kV electron microscope (JEM 200 A), thick (up to 0.4 μm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 μm thick each apex of the triangle was clearly blunted. This unique cross-sectional profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 μm to 0.3 μm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 μm thick. All of these observations and conclusions strongly support the model for the myosin filament proposed by Pepe (1966,1967). 相似文献
3.
Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer 总被引:3,自引:1,他引:2
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The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice. 相似文献
4.
The Z lattice in canine cardiac muscle 总被引:3,自引:3,他引:0
Filtered images of mammalian cardiac Z bands were reconstructed from optical diffraction patterns from electron micrographs. Reconstructed images from longitudinal sections show connecting filaments at each 38-nm axial repeat in an array consistent with cross-sectional data. Some reconstructed images from cross sections indicate two distinctly different optical diffraction patterns, one for each of two lattice forms (basket weave and small square). Other images are more complex and exhibit composite diffraction patterns. Thus, the two lattice forms co-exist, interconvert, or represent two different aspects of the same details within the lattice. Two three-dimensional models of the Z lattice are presented. Both include the following features: a double array of axial filaments spaced at 24 nm, successive layers of tetragonally arrayed connecting filaments, projected fourfold symmetry in cross section, and layers of connecting filaments spaced at intervals of 38 nm along the myofibril axis. Projected views of the models are compared to electron micrographs and optically reconstructed images of the Z lattice in successively thicker cross sections. The entire Z band is rarely a uniform lattice regardless of plane of section or section thickness. Optical reconstructions strongly suggest two types of variation in the lattice substructure: (a) in the arrangement of connecting filaments, and (b) in the arrangement of units added side-to-side to make larger myofilament bundles and/or end-to-end to make wider Z bands. We conclude that the regular arrangement of axial and connecting filaments generates a dynamic Z lattice. 相似文献
5.
We have used the method of three-dimensional image reconstruction of electron micrographs to analyse the structure of thin filaments and pure F-actin filaments decorated with myosin subfragment-1. To help improve on the earlier work of Moore et al. (1970), we have obtained all our data using minimal electron dose procedures to reduce radiation damage. Modifications in the specimen preparation have enabled us to process straight stretches of filament twice as long as any used in the earlier work, resulting in a corresponding improvement in the signal-to-noise ratio and the resolution. The results show significant changes in the density distribution in the region near the axis of the structure. Compared with the earlier model, the reconstructions show the presence of extra density close to the axis of the particle. We present a case for identifying actin with the density in this region, rather than with the density at higher radius previously designated as actin. This new assignment for the position of actin within the decorated filament structure leads to a radical change in the geometry of the model for myosin subfragment-lactin interaction. Furthermore, by comparing the features that we identify as actin with the reconstructed images of undecorated thin filaments published by Wakabayashi et al. (1975), we conclude that the polarity that has previously been assumed for the thin filament is incorrect. When the thin filament polarity is reversed, the position that tropomyosin is believed to occupy in the active state coincides with a weakly resolved feature in our reconstructions of decorated thin filaments. These findings, involving a reversal of thin filament polarity combined with the change in the geometry of myosin subfragment-1-actin interaction, allow a revised steric blocking model to be constructed. 相似文献
6.
Sartorius muscles from the green tree frog Hyla cerulea were set at variety of muscle lengths and fixed for electron microscopy using acrolein followed by osmium tetroxide. The sarcomere length, s, was determined in thick sections using laser-diffraction. The Z-disk lattice spacing, z, was measured in electron micrographs of thin sections from the same muscles. The Z-disk lattice was found to expand as sarcomere length decreased such that the quantity sz2 was constant at 1-05 X 10(6) nm3 for all sarcomere lengths in the range 1-9-2-9 mum. Thus, the sarcomere length dependency of the Z-disk lattice is similar to that of the myosin filament lattice. The density of thin filaments per unit cross section of fibril leaving the Z-disk is less than their density in the A band. Thus, fibrils have a smaller cross section in the I band, leaving more inter-fibrillar space there. This may explain why more mitochondria and lipid droplets are located in the I bands than in the A bands. It is suggested that the Z-disk may contributed to the short range elasticity of muscle fibres. 相似文献
7.
《Micron and Microscopica Acta》1984,15(4):221-226
A method is described which allows a three-dimensional object to be reconstructed from micrographs of serial thin sections using computer graphic techniques. The reconstructed object, which can be rotated three- dimensionally, is displayed on a colour visual display unit and the surface of the object is shaded in order that it can be observed to provide an illusion of a three-dimensional structure. Moreover, the technique makes it possible to represent an inner structure when seen through an outer one, also to observe other sectioned face views. The method as described here allows rapid visual evaluation of the results of three-dimensional reconstruction from serial thin sections when recorded with the aid of a light or an electron microscope. 相似文献
8.
Frank A. Pepe Francis T. Ashton Peter Dowben Murray Stewart 《Journal of molecular biology》1981,145(2):421-440
Improved fixation procedures have enabled substructure to be observed by electron microscopy in transverse sections of vertebrate skeletal muscle thick filaments as thin as 140 nm. Optical diffraction combined with digital autocorrelation analysis, focal series and tilting experiments have confirmed the presence of a regular substructure having a repeat near 4 nm and shown that it is highly unlikely to be an artifact associated with the electron microscope imaging system. The results obtained strongly suggest that the thick filament is constructed from a bundle of rod-like subfilaments arranged parallel to the thick filament axis to within less than a degree. This cannot easily be reconciled with the general theory of thick filament structure proposed by Squire (1973), but it is consistent with the model proposed by Pepe, 1966, Pepe, 1967. Optical diffraction of 140 nm thick serial transverse sections has also suggested a structural change along the length of the filament that is manifest by a variation in the proportion of filaments showing strong diffraction maxima in one, two or three directions. 相似文献
9.
The transforms of a large number of models of deoxygenated sickle hemoglobin fibers, related to that derived from image reconstruction of electron micrographs, have been calculated and compared with X-ray diffraction data of 15 A resolution. The model of the fiber, determined from the reconstructed image, is a helix consisting of 14 filaments that associate in a specific mode to form seven pairs, or protofilaments. Pairs were identified through the pattern of filament loss in partially disassembled fibers and by the separation between molecules, in adjacent filaments, of half a molecular diameter, along the fiber axis. An alternative mode of filament association can be derived also from the surface lattice of the reconstruction, which meets these criteria for the pairing of molecular filaments. Both pairing modes have been used in the search for structures whose transforms show the best agreement with the diffraction data. Models were generated by the systematic translation of six protofilaments, taken in symmetry related pairs, in steps of 3.5 A along the fiber axis relative to a fixed central protofilament. Each translation of a protofilament corresponds to a different fiber model, whose transform was compared with observed data. In all, over 11,000 transforms were calculated. Of all the models considered, three have been found whose residuals are minimal. At 30 A resolution, similar to that of electron micrographs, the model derived from image reconstruction and the three found through our search procedure are indistinguishable. At 15 A, however, the transforms of these models show better agreement with the observed data than the transform of the reconstructed image. Comparison of residuals shows that the model derived from the reconstructed image can be rejected with 99.5% probability relative to the model, with the same pairing scheme, found by our search procedures. The two other models, derived from the alternative pairing scheme, are also more credible than the reconstructed image, but at a lower confidence level. Each of our three models is equally acceptable. Their existence may reflect structural polymorphism of the fiber. 相似文献
10.
PEACHEY LD 《The Journal of biophysical and biochemical cytology》1958,4(3):233-242
Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mmicro; silver, 60 to 90 mmicro; gold, 90 to 150 mmicro; purple, 150 to 190 mmicro; blue, 190 to 240 mmicro; green, 240 to 280 mmicro; and yellow, 280 to 320 mmicro. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed. 相似文献
11.
By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different. 相似文献
12.
《The Journal of cell biology》1977,75(3):818-836
Optical diffraction patterns from electron micrographs of both longitudinal and cross sections of normal and anomalous canine cardiac Z bands have been compared. The data indicate that anomalous cardiac Z bands resembling nemaline rods are structurally related to Z bands in showing a repeating lattice common to both. In thin sections transverse to the myofibril axis, both electron micrographs and optical diffraction patterns of the Z structure reveal a square lattice of 24 nm. This lattice is simple at the edge of each I band and centered in the interior of the Z band, where two distinct lattice forms have been observed. In longitudinal sections, oblique filaments visible in the electron micrographs correspond to a 38-nm axial periodicity in diffraction patterns of both Z band and Z rod. We conclude that the Z rods will be useful for further analysis and reconstruction of the Z lattice by optical diffraction techniques. 相似文献
13.
Arrangement of filaments and cross-links in the bee flight muscle Z disk by image analysis of oblique sections 总被引:5,自引:4,他引:1
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Information from oblique thin sections and from three-dimensional reconstructions of tilted, transverse thin sections (Cheng, N., and J. F. Deatherage. 1989. J. Cell Biol. 108:1761-1774) has been combined to determine the three-dimensional structure of the honeybee flight muscle Z disk at 70-A resolution. The overall symmetry and structure of the Z disk and its relationship to the rest of the myofibril have been determined by tracing filaments and connecting elements on electron images of oblique sections which have been enhanced by a local crystallographic averaging technique. In the three-dimensional structure, the connecting density between actin filaments can be described as five compact, crystallographically nonequivalent domains. Features C1 and C2 are located on the transverse twofold rotation axes in the central plane of the Z disk. They are associated with the sides of actin filaments of opposite polarity. Features C3, C4, and C5 are present in two symmetry-related sets which are located on opposite sides of the central plane. C3 and C5 are each associated with two filaments of opposite polarity, interacting with the side of one filament and the end of the other filament. C3 and C5 may be involved in stabilizing actin filament ends inside the Z disk. The location of the threefold symmetric connection C4, relative to the thick filament of the adjacent sarcomere, is determined and its possible relationship to the C filament is considered. 相似文献
14.
The molecular packing of the subfilaments in muscle thick filaments has been investigated by electron microscopy. Thin (80-100 nm) transverse sections of vertebrate skeletal muscle were cut, and 129 electron microscope images of thick filaments from 15 different areas including seven to ten images in each area were analyzed by computer image processing. The transverse sections were limited to the portion of the filaments between the bare zone and the C-protein bearing region. Of the 129 images, six were discarded because they were structurally disrupted, 17 did not show evidence for the presence of subfilaments from the autocorrelation function, and four did not show evidence for three-fold rotational symmetry from the power spectrum. The remaining 102 filaments all showed evidence for three-fold rotational symmetry, consistent with other available evidence (Pepe, 1982). From the analysis of these images by rotational filtering, we have found that the vertebrate skeletal myosin filament is made up of nine subfilaments and that the image appears to have trigonal symmetry. Of these subfilaments, six are arranged with a center-to-center spacing of about 4 nm and the other three on the surface of the filament are distorted from this arrangement. Three additional densities, which together with the other nine, correspond to the pattern of 12 densities previously observed in more highly selected images (Stewart et al., 1981; Pepe and Drucker, 1972) were observed in 5% of the images. Another pattern of nine subfilaments peripherally arranged around the circumference of the filament was observed occasionally. This latter image may represent the organization of the subfilaments in the bare zone region of the filament, resulting from sampling of individual filaments displaced longitudinally relative to the other filaments in the A-band. 相似文献
15.
Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+. 总被引:1,自引:1,他引:0
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Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius. 相似文献
16.
《The Histochemical journal》1996,28(4):247-255
Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means
of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle
with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like
immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the
whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both
snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely
sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These
results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled
when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments
than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results
of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in
smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated
muscles. 相似文献
17.
Visualization of nucleosomes in thin sections by stereo electron microscopy 总被引:2,自引:1,他引:1
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《The Journal of cell biology》1980,87(3):833-836
Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding. 相似文献
18.
Murray Stewart Francis T. Ashton Robert Lieberson Frank A. Pepe 《Journal of molecular biology》1981,153(2):381-392
Computer image processing of electron micrographs has been employed to delineate the position of thick filament subunits in transverse sections of extensively crosslinked vertebrate skeletal muscle. Both back projection and rotational averaging methods indicate the presence of 12 subunits arranged on an approximately hexagonal lattice similar to that proposed by Pepe (1967). The spacing between subunits and the myosin content of the thick filament indicate that these subunits probably contain more than one myosin molecule and are most likely dimers. 相似文献
19.
Tarantula leg muscles in the relaxed state were rapidly frozen against a copper block cooled with liquid helium. Thin longitudinal sections of freeze-substituted specimens, both live and skinned, clearly showed the helical tracks of crossbridges on the surface of the myosin filaments, which are not preserved by conventional fixation. Fourier transforms of selected filaments showed a myosin layer line pattern, similar to that observed in X-ray diffraction patterns of intact tarantula muscle, extending to the sixth order of the 43.5 nm X-ray repeat. The phases of corresponding reflections were similar on the two sides of the meridian on the first layer line, and the crossbridge arrangement showed a line of mirror symmetry running down the center of the filament. These observations show that the number of helices (N) is even, in agreement with N = 4 determined from image analysis of negatively stained, isolated tarantula filaments (Crowther et al., J. Mol. Biol. 184, 429-439, 1985). Filtered images showed clear detail of the crossbridge helices and were similar to filtered images of negatively stained, isolated thick filaments. Thus, rapid freezing combined with freeze-substitution preserves the crossbridges in a three-dimensional arrangement approximating that occurring in vivo. 相似文献
20.
Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections. 总被引:2,自引:1,他引:1
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B L Trus A C Steven A W McDowall M Unser J Dubochet R J Podolsky 《Biophysical journal》1989,55(4):713-724
For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity. 相似文献